FIGURE 3.

Telethonin is constitutively phosphorylated in ventricular myocytes and tissue. A, Phos-tag SDS-PAGE and immunoblot analysis using the monoclonal anti-telethonin antibody of samples from ARVM infected with AdV:EGFP or AdV:wtPKD and stimulated with 100 nm ET-1 or 10 μm PE (with 1 μm atenolol) for 10 min. The same samples were subjected to standard SDS-PAGE and immunoblot (IB) analysis using the same antibody, and membranes were stained after use with Coomassie, as indicated. 2P, dual-phosphorylated. B, Phos-tag SDS-PAGE and immunoblot analysis using the monoclonal anti-telethonin antibody of samples from uninfected ARVM incubated (30 min at 30 °C) in the absence (-) or presence (+) of λPPase. The same samples were subjected to standard SDS-PAGE and immunoblot analysis using the same antibody, and membranes were stained after use with Coomassie as indicated. 0P, non-phosphorylated. C and D, Phos-tag SDS-PAGE and immunoblot analysis using the monoclonal anti-telethonin antibody of homogenates from rat (C) or mouse (D) ventricular tissues from three separate hearts in each case (left panels). Data are also shown for rat (C) and mouse (D) ventricular samples incubated (30 min at 30 °C) in the absence (-) or presence (+) of λPPase (right panels). The same samples were subjected to standard SDS-PAGE and immunoblot analysis using the same antibody, and membranes were stained after use with Coomassie as indicated. In A–D, recombinant His₆-tagged WT telethonin (100 pmol) phosphorylated in vitro by PKD_cat for 3 min was included as an internal standard in the gels. E, summary data from Fourier transform high-energy collisional dissociation MS sequencing of a bis-phosphorylated telethonin phosphopeptide fragment from mouse myocardium (all masses in Dalton). A mass difference from the expected mass of ∼80 Da (equivalent to -PO₃) is observed at both Ser-157 and Ser-161, indicating phosphorylation at these residues. aa, amino acid.